The long-term objective is the study of the roles that the chemical senses play in mammalian reproduction. The model system is the Asian elephant, Elephas maximus, a mammal of high intelligence with a well-developed, sophisticated social system. In elephants, chemosensory input has a definitive influence on pre-mating and mating behavior. For several weeks prior to ovulation, the urine of female Asian elephants contains extractable amounts of chemical compounds that elicit a high frequency of flehmen responses from bulls as an integral part of the mating scenario. A compound never before described in mammals, indolo[2,1-b]-quinazoline-6,12-dione (tryptanthrine), was identified as composing most of the active fraction purified from pre-ovulatory urine. The authentic synthetic compound was not bioactive. The hypothesis is that another component in the active preparation is responsible for the bioactivity. Accordingly, the principal objectives of the proposed investigation are: (1) Separate and purify the active pheromone from tryptanthrine, primarily by modifications of our well-established HPLC fractionation protocol. (2) Structurally Identify the bioactive compound, by first determining its mass, elemental composition, and possible radioimmunological activity, then chemically and structurally identifying it by field desorption mass spectrometry, electron ionization mass spectrometry, ultraviolet spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectrometry. (3) Synthesize and demonstrate the biological activity of this authentic compound. A proven bioassay involving the flehmen response by bulls will be used to guide the purification and assess the bioactivity of the synthetic compound. Collaborators Drs. Terry Lee, Beckman Research Institute, David Hess, Oregon Regional Primate Center, G. Doyle Daves, Rensselaer Polytechnic Institute, and Michael Schmidt, Washington Park Zoo, are essential to the success of this project. Drs. Lee and Daves offer access to state-of-the-art tools for structural analyses and expertise in structural identification. Since the isolated pheromone will probably be unusual, Dr. Daves is prepared to synthesize such a molecule for bioassay. Dr. Hess will provide comparative endocrinology expertise, and Dr. Schmidt directs animal care. Molecular and structural identification of the pheromone involved in sexual reproduction in the Asian elephant will make possible a more precise assessment of its role in reproductive physiology and chemical communication involving the vomeronasal organ (VNO). The VNO system projects selectively to the amygdaloid and basal forebrain structures which regulate sexual behavior and gonadotropin secretion, A single specific compound with known behavioral and biological effects will be immensely valuable for studying chemosensory signal transduction mechanisms using the VNO model system, especially when correlated with new ultrastructural information on VNO receptor cells and unusual receptors in the trunk tip finger. In addition, pharmacological studies with an active authentic compound will offer insights into pheromone-hormone interrelationships in males and females and into possible sources of mammalian sexual pheromones.